Genome editing is a powerful technology for genetic manipulation and modification. A recently developed genome modification technology utilizes the bacterial clusters of regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9), an RNA-guided DNA endonuclease, to induce a specific double-stranded break (DSB) at DNA target sites comprising a 3-nucleotide (nt) protospacer adjacent motif (PAM) and a 20-bp sequence complementary, to the 5′ end of a CRISPR guide RNA (gRNA) bound by Cas9. The guide RNA-Cas9 complex identifies and base pairs with its cognate DNA target sequence, resulting in target cleavage to form a DSB (FIG. 1). It has been observed that the CRISPR-Cas9 system tolerates a limited extent of base-pair mismatching between the DNA substrate and the 20-nt guide sequence of the guide RNA, which results in undesired off-target DNA cleavages (Carroll, D. (2013) Nat. Biotechnol., 31, 807-9).
As formation of off-target mutations is of profound concern to developers of genome editing technologies, three different strategies have been devised to improve the specificity of CRISPR-Cas9 genome modification (FIGS. 2A-2C). These strategies all reduce, but do not abolish, off-target DNA cleavage at a cost of diminishing the on-target cleavage activity with respect to the comparable unmodified CRISPR:Cas9 guided nuclease system.
The first strategy involves a Cas9 mutant (Cas9n; also known as Cas9-D10A) that cleaves and nicks just one strand of the target DNA. Two distinct guide RNA sequences are designed such that Cas9n creates offset nicks on opposite strands, thereby creating a DSB in the DNA target region (FIG. 2A). A variation of this strategy uses two different Cas9 mutants, each recognizing one of the two strands. Compared to the unmodified CRISPR-Cas9 system, this double nickase strategy requires two adjacent targetable DNA sequences and has lower on-target modification activity (Ran et al. (2013) Cell, 154, 1380-9). Additionally, although specificity is increased, there is still significant off-target activity, and single-stranded DNA nicks are weakly mutagenic (Mali et al., (2013) Science, 339, 823-6).
A second strategy employs cleavage-deactivated Cas9 (dCas9) fused to a dimeric-dependent FokI nuclease domain (FokI-dCas9) with two distinct guide RNAs specifying the DNA target site (FIG. 2B). Although this strategy demonstrated greater specificity than that of the paired nickase strategy, it showed further reduction in on-target cleavage efficiency as compared to the original CRISPR-Cas9 nuclease system using one or the other guide RNA of each pair (Tsai et al., (2014) Nat. Biotechnol., 32, 569-76). Additionally, as with the paired nickase strategy, the FokI-dependent approach is limited by its requirement for two adjacent targetable DNA sequences, potentially restricting its utility and versatility in genome modification applications.
In a third strategy, improvement in the specificity of CRISPR-Cas9 cleavage was achieved by truncating the 20-nt guide sequence by 2 or 3 nucleotides at its 5′ end to generate truncated guide RNAs (tru-RGNs, Fu et al., (2014) Nat. Biotechnol., 32, 279-84). Comparing the DNA cleavage activity of full-length guide RNA and tru-RGNs targeting a few different genomic sequences, it was demonstrated that off-target cleavage activity can be reduced with tru-RGNs. However, for some DNA target sequences, off-target activities of tru-RGNs were still significant and even elevated in one instance relative to off-target activities of Cas9 complexed with full-length guide RNA. Additional truncation of the 5′ end generally reduces on-target guide RNA-Cas9 activity, reducing it to background levels when the DNA-pairing sequence is truncated to 15 nt or shorter.
Thus, there is a need for reagents and methods for increasing specificity and efficiency of RNA-guided genome editing.